Gene therapy to promote thromboresistance: local overexpression of tissue plasminogen activator to prevent arterial thrombosis in an in vivo rabbit model.

Tissue-type plasminogen activator (tPA) catalyzes the rate-limiting initial step in the fibrinolytic cascade. Systemic infusion of tPA has become the standard of care for acute myocardial infarction. However, even the relatively short-duration protocols currently employed have encountered significant hemorrhagic complications, as well as complications from rebound thrombosis. Gene therapy offers a method of local high-level tPA expression over a prolonged time period to avoid both systemic hemorrhage and local rebound thrombosis. To examine the impact of local tPA overexpression, an adenoviral vector expressing tPA was created. The construct was characterized functionally in vitro, and the function of the vector was confirmed in vivo by delivery to the rabbit common femoral artery. Systemic coagulation parameters were not perturbed at any of the doses examined. The impact of local overexpression of tPA on in vivo thrombus formation was examined subsequently in a stasis/injury model of arterial thrombosis. The construct effectively prevented arterial thrombosis in treated animals, whereas viral and nonviral controls typically developed occluding thrombi. This construct thus offers a viable technique for promoting a locally thromboresistant small-caliber artery.

Gene therapy for plastic and reconstructive surgery.

In the future, gene therapy will become the standard treatment for enhancing wound healing and nerve and muscle regeneration and for preventing or treating vessel thrombosis, areas critical to the plastic surgeon and the patient. This review has focused on factors that are either currently used in a clinical situation or have the promise of being developed for clinical use. The role of gene therapy applied to these areas will be to locally place therapeutic genes at the site of injury or surgical repair, which will replace the need to systemically administer therapeutic agents that may have toxic effects, and to express factors in low abundance but at effective and safe levels. This strategy is to produce enough agent to have a therapeutic effect. The use of tissue-specific promoters will be beneficial in localizing the production of agents to certain cell types. Gene therapeutic approaches used in plastic surgery may have a significant impact on the health care system by increasing efficiency of treatment and reducing health care costs. The accelerated pace of basic research in gene therapy will result in clinical applications in the near future.

Monoclonal antibody BLT-1, specific for the bovine homologue of CD5, reacts with the majority of mature T cells, a subpopulation of B cells and stimulates T cell proliferation.

Monoclonal antibody (MAb) BLT-1, an IgG2a with kappa light chains, reacted strongly with 21% of bovine thymocytes, weakly with 15% of thymocytes, with a subpopulation of peripheral blood B cells that also expressed CD20 and with peripheral blood T cells. Practically all of the reactive thymocytes were of a large cell subpopulation. By immunoprecipitation, BLT-1 was shown to recognize a membrane molecule with a molecular mass of 67 kDa. In competitive assays for lymphocyte surface binding, BLT-1 and MAb CC-29 (which had been shown previously to react with bovine CD5) blocked one another, indicating that the epitopes recognized were identical or extensively overlapping. In contrast, another CD-5-reactive MAb, CC-17, did not block BLT-1 reactivity with lymphocytes although the reactivity of CC-17 was blocked by BLT-1, suggesting partial overlap of the epitopes or steric hindrance by BLT-1 but not by CC-17. BLT-1 was able to induce proliferation of bovine lymphocytes in culture alone if monocytes were present or in the absence of monocytes synergized with PMA. The results indicate that BLT-1 recognizes an epitope of the bovine homologue of CD5 and that perturbation of the epitope by MAb binding results in signal transduction to bovine lymphocytes.

Regulation of tyrosine hydroxylase gene expression in IMR-32 neuroblastoma cells by basic fibroblast growth factor and ciliary neurotrophic factor.

The actions of basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF) on tyrosine hydroxylase (TH) gene expression were studied using IMR-32 neuroblastoma cells. Treatment of these cells with bFGF for 3 days induced the expression of detectable levels of immunoreactive TH protein and TH mRNA. In contrast, CNTF did not affect TH expression unless bFGF was present. In the presence of saturating amounts of bFGF, CNTF increased TH protein and mRNA levels of TH two-to threefold over those found in bFGF-treated cultures. The effects of CNTF on TH expression diminished with increasing culture time, and after 6 days of incubation CNTF no longer enhanced TH levels. The requirement for bFGF as cofactor in the effects of CNTF on TH was specific, as CNTF did not affect TH when it was coadministered with 8-(4-chlorophenylthio)-cyclic AMP, another agent that stimulates TH development in this cell line, and bFGF was not required for CNTF to stimulate the development of choline acetyltransferase. Moreover, cotreatment with bFGF reduced the ability of CNTF to enhance choline acetyltransferase. These results demonstrate that bFGF and CNTF can enhance expression of TH and that bFGF can modify the effects of CNTF on neurotransmitter phenotype.

Preparation and affinity purification of a novel, biologically active, CNTF fusion protein.

A biologically active fusion protein comprising a short hydrophilic leader peptide fused to the N-terminus of rat CNTF was generated using commercially available materials. The coding region for rat CNTF was sub-cloned into the pFLAG-1 vector and transfected into the JM 109 strain of E. coli. The transfected cells expressed high levels of the fusion protein (FLAG-CNTF) following induction by isopropyl beta-D-thiogalactoside (IPTG). FLAG-CNTF is expressed as insoluble material that was resolubilized by extraction with guanidine hydrochloride and purified by immuno-affinity chromatography. Analysis of the purified material by reverse phase HPLC and Western blot analysis indicated that FLAG-CNTF was composed of two closely related species that are greater than 99% pure after affinity chromatography. The purified FLAG-CNTF migrated as a 27 KD doublet on SDS polyacrylamide gel electrophoresis. Both bands of the doublet were shown to contain the FLAG peptide and CNTF by Western blot analysis, and amino acid sequence analysis demonstrated a single amino acid sequence corresponding to FLAG peptide and the N-terminus of CNTF. The purified fusion protein was tested for biological activity using the IMR-32 human neuroblastoma cell line. Treatment of IMR-32 cells with FLAG-CNTF increased the level of choline acetyltransferase (ChAT) in these cells 2-3-fold over that of control cells in a dose dependent manner. A direct comparison of the effects of FLAG-CNTF and recombinant human CNTF on IMR-32 ChAT activity showed that both factors exhibited similar potencies.(ABSTRACT TRUNCATED AT 250 WORDS)

Temporal and spatial expression of ciliary neurotrophic factor after peripheral nerve injury.

Schwann cells in the intact sciatic nerve express high amounts of ciliary neurotrophic factor (CNTF), but 7 days after injury to the nerve expression dramatically decreases. To determine whether this change occurs only in the region of the injury or throughout the whole nerve we examined the spatial and temporal expression of CNTF after a crush injury. One day after injury the amount of CNTF mRNA and protein decreased within the first 4 mm distal to the crush site. This decrease progressed in a centrifugal manner distally until mRNA and protein were scarcely detectable by 7 days. In nerve proximal to the crush site CNTF expression decreased slightly and was still detectable at all sample times. During regeneration CNTF expression remained very low up to 14 days after injury. By 30 days mRNA and protein were detectable and by 60 days CNTF protein was present at normal amounts. Immunohistochemical analysis of normal nerve revealed CNTF localized in outer portion of the cytoplasm of myelin-forming Schwann cells. Three days after injury CNTF coalesced with pockets of cytoplasm in the Schwann cell and by 5 days was barely detectable. Positive staining remained in proximal segments where little or no degeneration occurred. These results demonstrate that CNTF expression in Schwann cells is synchronized with their functional state. CNTF expression decreases with demyelination during Wallerian degeneration and returns to normal following remyelination during regeneration. These findings also suggest that CNTF expression requires intact axon-Schwann cell interactions.

Differential effects of neurotrophic factors on neurotransmitter development in the IMR-32 human neuroblastoma cell line.

The human neuroblastoma cell line IMR-32 exhibits both cholinergic and adrenergic properties. We have used IMR-32 cells to study the effects of CDF (CAT development factor) and bFGF (basic fibroblast growth factor) on the development of neurotransmitter properties. CDF treatment increases CAT activity in a dose-dependent manner, independent of cell density. Time course studies show that there is a threefold increase in the specific CAT activity in IMR-32 cells treated with CDF for 6 d. CDF does not, however, affect the level of tyrosine hydroxylase (TH) activity, or the rate of cell proliferation. bFGF, on the other hand, induces TH activity and decreases CAT activity in a dose-dependent manner. bFGF's effect on TH is enhanced by increasing cell density, while its reduction of specific CAT activity is independent of cell density. Time course studies show a 30-fold increase in TH activity per cell and a threefold decrease in CAT activity per cell, after treatment with bFGF for 6 d. In contrast to the effects of CDF, bFGF enhances cell proliferation in IMR-32 cells. Double-labeled immunofluorescence studies showed that 95% of the cells stain for CAT and 65% stain for TH following treatment with CDF and bFGF, respectively. When these factors are combined, approximately 75% of the cells express both CAT and TH, demonstrating that IMR-32 cells are bipotential with regard to neurotransmitter-associated enzyme expression. We also show that insulin-like growth factor I and NGF selectively induce CAT activity and cell proliferation, respectively, whereas epidermal growth factor has no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Peripheral nerve injury down-regulates CNTF expression in adult rat sciatic nerves.

Ciliary neurotrophic factor (CNTF) is a 200-amino acid protein expressed in high concentrations by peripheral nerves and is thought to be important for the survival and regeneration of injured motoneurons (Lin et al., J Biol Chem 265:8942-8947, 1990). To better understand CNTF's role in nerve injury we have characterized the effects of crush injury on the expression of CNTF in adult rat sciatic nerves using specific antibody and RNA probes. Following a crush injury, both the protein and mRNA levels undergo pronounced decreases distal to the crush. These changes in CNTF expression were qualitatively distinct from changes in the expression of the low-affinity NGF receptor (p75NGFR), which increases following crush. Thus, the changes in CNTF levels do not reflect an overall down-regulation of mRNA during degeneration, and are inconsistent with the proposed role of CNTF in neuronal injury, since its levels are decreasing at the same time as the requirement for neurotrophic support is increasing.

Changes in subpopulations of lymphocytes in peripheral blood, and supramammary and prescapular lymph nodes of cows with mastitis and normal cows.

Direct immunofluorescence (IF) and indirect IF techniques were employed to analyze the distribution of B and T lymphocyte populations in peripheral blood, and in supramammary (draining), and prescapular (non-draining) lymph nodes of cows with mastitis and normal cows. In the peripheral blood there was a significant decrease in the percent and absolute number of B lymphocytes in mastitic cows (n = 29; 17.1 +/- 10.2%; 3.4 +/- 2.7 X 10(5) cells/ml) as compared to normal cows (n = 38; 25.2 +/- 7.8%; 9.3 +/- 5.4 X 10(5) cells/ml). The percent T lymphocyte count in mastitic cows (71.2 +/- 7.1%) was slightly increased over that of normals (65.8 +/- 7.2%), although the absolute number of T lymphocytes was decreased in mastitic cows (1.49 +/- 0.91 X 10(6) cells/ml vs. 2.47 +/- 1.28 X 10(6) cells/ml). In the prescapular lymph node the percent of B lymphocytes, but not T or "null lymphocytes", decreased significantly in mastitic cows as compared to that of normals. The decrease, i.e. 32%, paralleled the 32.1% decrease found in peripheral blood B lymphocytes. In contrast, in the supramammary lymph node of mastitic cows, the percent B lymphocytes increased over that of normals (35.1 +/- 2.0% vs. 20.4 +/- 9.4%), whereas the percent T lymphocytes decreased to 54.5 +/- 2.8% compared to 70.7 +/- 3.5% in normal cows. There was no significant change in percent "null lymphocytes". The weight of prescapular lymph nodes did not change in mastitic cows when compared to that of normals. As a result, the estimated number of B lymphocytes, but not of T and "null lymphocytes", decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Separation and identification of bovine lymphocyte populations.

Various methods for separation of lymphocyte populations have been modified and adapted for use in isolating and identifying bovine lymphocytes. Ficoll diatrizoate (F-D) with a specific density of 1.084 was found to be superior to those with densities of 1.080 and 1.077 which were developed originally for the mouse and human mononuclear cells, respectively. F-D with a density of 1.084 attained a lymphocyte (absolute number) recovery rate of 92% whereas those with densities of 1.080 and 1.077 yielded 81% and 71% recovery rate of lymphocytes, respectively. Subsequent separation of T lymphocytes was achieved best by nylon wool column whereas separation of B lymphocytes was attained best by complement-mediated depletion of T lymphocytes with the T lymphocyte specific monoclonal antibody (MAb), BLT-1. The former yielded 95 +/- 3% T lymphocytes with 47 +/- 9% recovery rate, and the latter gave 96 +/- 3% B lymphocytes with 71 +/- 9% recovery rate. In comparison, direct panning of F-D gradient separated mononuclear cells with goat anti-bovine IgG coated plates yielded 80% B lymphocytes with 31% recovery rate and indirect panning of MAb BLT-1 treated F-D gradient-separated mononuclear cells with goat anti-mouse IgG coated plates yielded 89% T lymphocytes with 35% recovery rate.

Production and characterization of a bovine T cell-specific monoclonal antibody identifying a mature differentiation antigen.

A monoclonal antibody (MAb), BLT-1, with specificity for bovine mature T cells was prepared by somatic cell hybridization of myeloma NS-1 and spleen cells from BALB/c mice hyperimmunized with bovine T lymphocytes. The MAb reacted with over 92% of nylon wool-nonadherent lymphocytes (T cells) but not with nylon wool-adherent EAC-positive lymphocytes (B cells) in the indirect immunofluorescence assay. It is an IgM, with kappa-light chains, which fixed complement well and killed over 95% of mature T cells in complement-mediated cytotoxicity assays. It reacted with the same proportions of peripheral lymphoid cells (peripheral blood, lymph nodes, and spleen) as the polyclonal goat anti-bovine thymocyte serum (GABTS), but only with 25% of GABTS-positive thymocytes. Immunoperoxidase staining of frozen tissue sections showed that the BLT-1-positive cells were located in the medulla of the thymus and in the T lymphocyte areas of lymph nodes. Western immunoblotting assays showed that the BLT-1-reactive membrane antigen is a 22,000 m.w. protein which was inducible in bovine thymocytes with bovine thymic hormones, thymosin fraction 5, thymosin alpha 1, and thymopentin ORF-18150, indicating that it is a mature T lymphocyte differentiation antigen. The thymosin alpha 1 and thymopentin were found to show additive effects on mature T cell antigen expression by bovine thymocytes.

Effects of horse and fetal calf serum on the expression of tumor-associated antigen and tumorigenicity of L5178Y leukemia/lymphoma cells.

A tumor antigen (TA) associated with murine leukemia-lymphoma L5178Y cells has been identified by the enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IIF) techniques. The antigen was present in both non-solubilized and 0.5% NP-40 solubilized membrane extracts. Rabbit anti-L5178Y lymphoma serum (RALS), extensively absorbed with normal mouse tissues, identified TA in extracts of L5178Y lymphoma and L5178Y leukemia cells grown in horse serum (L5178Y/HS), but not in extracts of L5178Y cells grown in fetal calf serum (L5178Y/FCS). Similarly, absorbed rabbit anti-L5178Y/HS serum specifically reacted with extracts of lymphoma and L5178Y/HS but not with L5178Y/FCS cells. Membrane IIF showed positive reactivity in 88% of lymphoma and 73% of L5178Y/HS cells, whereas splenic lymphocytes and L5178Y/FCS cells were negative. Goat anti-AKR virus serum reacted with soluble extracts of lymphoma, L5178Y/HS, and L5178Y/FCS as well as with normal DBA/2 tissues in the ELISA. However, goat anti-AKR virus serum did not block the reactivity of RALS to lymphoma in the blocking ELISA (BELISA). Expression of TA, but not murine leukemia viral antigen(s), was correlated with the in-vivo tumorigenicity of the L5178Y cells. The antigenic activity of lymphoma extract was reduced by incubation for 1 h at 56 and 65 degrees C, by trypsin digestion, and by exposure to pH 2.8 or 11.0 for 1 h. The antigen, sequentially purified by gel filtration and Lentil-lectin affinity chromatography, was a glycoprotein, with a molecular weight of approx. 64,000 daltons, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

Identification and quantitation of bovine T cell antigen with enzyme-linked immunosorbent assay (ELISA).

The enzyme-linked immunosorbent assay (ELISA) was used to identify and quantitate T cell antigen(s) in bovine thymocytes and peripheral blood T cells. The ELISA, employing non-ionic detergent-solubilized lymphocyte membranes as antigen, and goat anti-bovine thymocyte serum (GABTS) and horseradish peroxidase-conjugated rabbit anti-goat immunoglobulin (PORAG) as direct and indirect reactants, respectively, showed that thymocytes and peripheral blood T cells separated by erythrocyte-antibody-complement (EAC) rosetting technique had high concentrations of T cell antigen. In contrast, bone marrow cells and EAC-positive peripheral blood B cells had minimal T cell antigen. 92.0 +/- 3.5% of EAC-positive bovine lymphocytes were found to be B cells and 89.5 +/- 5.8% of EAC-negative lymphocytes T cells, as determined by SmIg and anti-T cell immunofluorescence (IF) techniques. In both IF and ELISA, pre-absorption of rabbit anti-goat IgG serum was needed to obviate cross-reactions with bovine B cells. A competitive ELISA developed for quantitating T cell antigens, showed that peripheral blood T cells contained only 46% of the antigen present in thymocytes.